Christopher D. Porada

Adult mesenchymal stem cells: a pluripotent population with multiple applications.

Mesenchymal stem cells (MSCs) have been isolated not only from bone marrow, but also from many other tissues such as adipose tissue, skeletal muscle, liver, brain and pancreas. Because MSC were found to have the ability to differentiate into cells of multiple organs and systems such as bone, fat, cartilage, muscle, neurons, hepatocytes and insulin-producing cells, MSCs have generated a great deal of interest for their potential use in regenerative medicine and tissue engineering. Furthermore, given the ease of their isolation and their extensive expansion rate and differentiation potential, mesenchymal stem cells are among the first stem cell types that have a great potential to be introduced in the clinic. Finally, mesenchymal stem cells seem to be not only hypoimmunogenic and thus be suitable for allogeneic transplantation, but they are also able to produce immunosuppression upon transplantation. In this review we summarize the latest research in the use of mesenchymal stem cells in transplantation for generalized diseases, local implantation for local tissue defects, and as a vehicle for genes in gene therapy protocols.

Mesenchymal stem cells as therapeutics and vehicles for gene and drug delivery.

Mesenchymal stem cells (MSCs) possess a set of several fairly unique properties which make them ideally suited both for cellular therapies/regenerative medicine, and as vehicles for gene and drug delivery. These include: 1) relative ease of isolation; 2) the ability to differentiate into a wide variety of seemingly functional cell types of both mesenchymal and non-mesenchymal origin; 3) the ability to be extensively expanded in culture without a loss of differentiative capacity; 4) they are not only hypoimmunogenic, but they produce immunosuppression upon transplantation; 5) their pronounced anti-inflammatory properties; and 6) their ability to home to damaged tissues, tumors, and metastases following in vivo administration. In this review, we summarize the latest research in the use of mesenchymal stem cells in regenerative medicine, as immunomodulatory/anti-inflammatory agents, and as vehicles for transferring both therapeutic genes in genetic disease and genes designed to destroy malignant cells.

Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep

Alternative methods to whole liver transplantation require a suitable cell that can be expanded to obtain sufficient numbers required for successful transplantation while maintaining the ability to differentiate into hepatocytes. Mesenchymal stem cells (MSCs) possess several advantageous characteristics for cell‐based therapy and have been shown to be able to differentiate into hepatocytes. Thus, we investigated whether the intrahepatic delivery of human MSCs is a safe and effective method for generating human hepatocytes and whether the route of administration influences the levels of donor‐derived hepatocytes and their pattern of distribution throughout the parenchyma of the recipients liver. Human clonally derived MSCs were transplanted by an intraperitoneal (n = 6) or intrahepatic (n = 6) route into preimmune fetal sheep. The animals were analyzed 56–70 days after transplantation by immunohistochemistry, enzyme‐linked immunosorbent assay, and flow cytometry. The intrahepatic injection of human MSCs was safe and resulted in more efficient generation of hepatocytes (12.5% ± 3.5% versus 2.6% ± 0.4%). The animals that received an intrahepatic injection exhibited a widespread distribution of hepatocytes throughout the liver parenchyma, whereas an intraperitoneal injection resulted in a preferential periportal distribution of human hepatocytes that produced higher amounts of albumin. Furthermore, hepatocytes were generated from MSCs without the need to first migrate/lodge to the bone marrow and give rise to hematopoietic cells. Conclusion: Our studies provide evidence that MSCs are a valuable source of cells for liver repair and regeneration and that, by the alteration of the site of injection, the generation of hepatocytes occurs in different hepatic zones, suggesting that a combined transplantation approach may be necessary to successfully repopulate the liver with these cells. (HEPATOLOGY 2007.)

Human Mesenchymal Stem Cells Form Purkinje Fibers in Fetal Sheep Heart

Background—We have investigated the usefulness of a model of cardiac development in a large mammal, sheep, for studies of engraftment of human stem cells in the heart. Methods and Results—Adult and fetal human mesenchymal stem cells were injected intraperitoneally into sheep fetuses in utero. Hearts at late fetal development were analyzed for engraftment of human cells. The majority of the engrafted cells of human origin formed segments of Purkinje fibers containing exclusively human cells. There were no differences in engraftment of human mesenchymal stem cells from adult bone marrow, fetal brain, and fetal liver. On average, 43.2% of the total Purkinje fibers in random areas (n=11) of both ventricles were of human origin. In contrast, ≈0.01% of cardiomyocytes were of human origin. Conclusions—Human mesenchymal stem cells preferentially engraft at high levels in the ventricular conduction system during fetal development in sheep. These findings raise the possibility that stem cells contribute to normal development of the fetal heart.

Differentiative potential of human metanephric mesenchymal cells

OBJECTIVE To evaluate the ability of mesenchymal cells derived from nonhematopoietic organs to form blood and other tissues in vitro and in vivo. MATERIALS AND METHODS Because of its mesodermic derivation, human fetal kidney was used as a source of mesenchymal cells. Two populations of kidney cells were studied at a nonclonal level: a crude preparation, and an adherent fraction that was derived from the first by propagation in vitro (MNMC). Both populations were transplanted into sheep fetuses and analyzed at intervals for the presence of human cells in different organs by flow cytometry, PCR, immunohistochemistry, and in situ hybridization. Secondary transplantation studies were performed using human hematopoietic cells obtained from the bone marrow (BM) of primary recipients. RESULTS MNMC were Thy-1(+), CD51(+), CD44(+), CD45(-), and vimentin(+), a phenotype consistent with that of metanephric mesenchyme. The crude population displayed the same phenotype but was contaminated with 0.4% CD34(+)CD45(+) cells. Cells with hepatocyte-like morphology and phenotype were obtained from the MNMC after culture in specific inducing media. After transplantation, both populations of cells produced multilineage hematopoietic engraftment and gave rise to CD34(+) cells. Successful hematopoietic engraftment in secondary recipients demonstrated the generation of long-term engrafting hematopoietic stem cells from MNMC. PCR analysis confirmed human hematopoietic engraftment and revealed that human cells were also present within other organs. Liver sections of transplanted animals contained human albumin-producing hepatocyte-like cells. CONCLUSION A human metanephric mesenchymal cell population simultaneously gave rise to human blood and liver-like cells, suggesting that mesenchymal cells may represent a broad population of putative stem cells in multiple adult organs.

Bone marrow stem cells and liver regeneration

Development of new approaches to treat patients with hepatic diseases that can eliminate the need for liver transplantation is imperative. Use of cell therapy as a means of repopulating the liver has several advantages over whole-organ transplantation because it would be less invasive, less immunogenic, and would allow the use, in some instances, of autologous-derived cells. Stem/progenitor cells that would be ideal for liver repopulation would need to have characteristics such as availability and ease of isolation, the ability to be expanded in vitro, ensuring adequate numbers of cells, susceptibility to modification by viral vector transduction/genetic recombination, to correct any underlying genetic defects, and the ability of restoring liver function following transplantation. Bone marrow-derived stem cells, such as hematopoietic, mesenchymal and endothelial progenitor cells possess some or most of these characteristics, making them ideal candidates for liver regenerative therapies. Here, we will summarize the ability of each of these stem cell populations to give rise to functional hepatic elements that could mediate repair in patients with liver damage/disease.

In utero transfer and expression of exogenous genes in sheep

OBJECTIVE We have previously reported that directly injecting low-titer retroviral vector supernatant into pre-immune sheep fetuses resulted in the transfer and long-term expression of the bacterial NeoR gene within the hematopoietic system of these animals for over 5 years. In the present studies, we investigated whether using a higher titer vector would enable more efficient transduction and expression of the transgenes within the hematopoetic cells in sheep injected in utero. MATERIALS AND METHODS Sixteen pre-immune sheep fetuses were injected intraperitoneally with the G1nBgSvNa8.1 helper-free retroviral vector supernatant encoding the bacterial NeoR and LacZ genes (titer: 1x10(7) cfu/mL). RESULTS Over the 2-year time course of these studies, the presence and expression of the NeoR and LacZ genes were demonstrated in 12 of the 14 animals evaluated by several immunological and biochemical methods. Seven of the 12 sheep examined by flow cytometric analysis contained > or =6% transduced peripheral blood lymphocytes. Vector distribution was widespread without any detectable pathology. Importantly, PCR analyses and breeding experiments demonstrated that the germ line was not altered. CONCLUSIONS These studies confirmed that direct injection of an engineered retrovirus is a feasible means of safely delivering foreign genes into a developing fetus and thus achieving long-term expression of the transgenes within the recipients hematopoietic cells. Furthermore, expression of the NeoR gene from these studies was higher than that reported in our previous study in which a lower titer vector was used.

Engraftment and Multilineage Expression of Human Bone Marrow CD34− Cells In Vivoa

Abstract: The fetal sheep competitive engraftment model of human hematopoietic stem cells (HSC) was used to evaluate the in vivo engraftment potential of human bone marrow CD34− Lin− cells. Transplantation of CD34− Lin− cells into primary hosts resulted in the long‐term (>1 year) engraftment and multilineage donor cell/progenitor expression with production of significant numbers of CD34+ cells. Secondary transplantation and limiting dilution studies confirmed the presence in human CD34− fraction of HSC with in vivo long‐term engraftment and multilineage differentiation potentials.

The effect of low-frequency electromagnetic field on human bone marrow stem/progenitor cell differentiation.

Human bone marrow stromal cells (hBMSCs, also known as bone marrow-derived mesenchymal stem cells) are a population of progenitor cells that contain a subset of skeletal stem cells (hSSCs), able to recreate cartilage, bone, stroma that supports hematopoiesis and marrow adipocytes. As such, they have become an important resource in developing strategies for regenerative medicine and tissue engineering due to their self-renewal and differentiation capabilities. The differentiation of SSCs/BMSCs is dependent on exposure to biophysical and biochemical stimuli that favor early and rapid activation of the in vivo tissue repair process. Exposure to exogenous stimuli such as an electromagnetic field (EMF) can promote differentiation of SSCs/BMSCs via ion dynamics and small signaling molecules. The plasma membrane is often considered to be the main target for EMF signals and most results point to an effect on the rate of ion or ligand binding due to a receptor site acting as a modulator of signaling cascades. Ion fluxes are closely involved in differentiation control as stem cells move and grow in specific directions to form tissues and organs. EMF affects numerous biological functions such as gene expression, cell fate, and cell differentiation, but will only induce these effects within a certain range of low frequencies as well as low amplitudes. EMF has been reported to be effective in the enhancement of osteogenesis and chondrogenesis of hSSCs/BMSCs with no documented negative effects. Studies show specific EMF frequencies enhance hSSC/BMSC adherence, proliferation, differentiation, and viability, all of which play a key role in the use of hSSCs/BMSCs for tissue engineering. While many EMF studies report significant enhancement of the differentiation process, results differ depending on the experimental and environmental conditions. Here we review how specific EMF parameters (frequency, intensity, and time of exposure) significantly regulate hSSC/BMSC differentiation in vitro. We discuss optimal conditions and parameters for effective hSSC/BMSC differentiation using EMF treatment in an in vivo setting, and how these can be translated to clinical trials.

Plasticity of human stem cells in the fetal sheep model of human stem cell transplantation.

Experimental models that allow the evaluation of the full potential of stem cells under normal physiological conditions and in the absence of genetic or injury-induced dysfunction would serve as valuable tools for the study of the mechanisms underlying stem cell differentiation. Ideally, such a model would also permit the robust formation of donor-derived tissue-specific cells. Because studies have shown that the differentiation of stem cells into cells of a different germinal layer is highly inefficient in the absence of selective pressure, it is very unlikely that a healthy adult animal can fulfill these requirements. In this review, we describe the advantages of the permissive aspects of the developing preimmune fetus in the early gestational age that led us to develop the sheep as a large-animal model of human stem cell plasticity.